Subpellicular and Flagellar Microtubules of Trypanosoma

Journal of Cell Science 110, 431-437 (1997) 431 Printed in Great Britain © The Company of Biologists Limited 1997 JCS3502 Subpellicular and flagellar microtubules of Trypanosoma brucei are extensively glutamylated André Schneider1, Uwe Plessmann2 and Klaus Weber2,* 1University of Fribourg, Institute of Zoology, Pérolles, CH 1700 Fribourg, Switzerland 2Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, PO Box 2841, D-37018, Goettingen, Germany *Author for correspondence SUMMARY To determine the spectrum of tubulin variants in cytoskel- residues 445 of α- and 435 of β-tubulin. The same sites are etons of Trypanosoma brucei carboxy-terminal fragments used in glutamylated tubulins of mammalian brain. No of α- and β-tubulin were isolated and characterized by tubulin variants based on polyglycylation are detected in sequencing and mass spectrometry. All variants arise by cytoskeletal preparations or in isolated flagella. Tubulin posttranslational modifications. We confirm the presence of specific incorporation of radioactive glutamate but not of tyrosinated and detyrosinated α-tubulin. Unexpectedly, but glycine is observed when protein biosynthesis is completely in line with its sequence, β-tubulin also occurs with and inhibited in Trypanosoma cells. Possible reasons for the without its carboxy-terminal tyrosine. Both tyrosinated absence of polyglycylated tubulins from the trypanosomal and detyrosinated α- and β-tubulins are extensively glu- axoneme are discussed. Finally we show that lysine 40 of tamylated. Unglutamylated tubulins are only trace compo- the flagellar α-tubulin is completely acetylated. nents of the cytoskeletal microtubules. The maximal numbers of glutamyl residues in the lateral chain are 15 and 6 for α- and β-tubulin, respectively. The oligoglutamyl Key words: Acetylation, Carboxypeptidase, Polyglutamylation, side chain is linked via an isopeptide bond to glutamic acid Posttranslational modification, Tyrosination INTRODUCTION and β-tubulin. In the case of polyglutamylation the position of this modified residue has been established for all brain tubulins Microtubules are involved in eukaryotic cell division, directed (Eddé et al., 1990; Alexander et al., 1991; Redeker et al., 1992; intracellular transport, the movement of cilia and flagella and Rüdiger et al., 1992; Mary et al., 1994). While the other they also influence the dynamic organisation of cellular mor- tubulin-specific PTMs first emerged in animal cells and tissues phology. The αβ-tubulin heterodimer, the structural unit of the polyglycination was first documented in the ciliary axonemal microtubules, is the target of a number of posttranslational microtubules of the ciliate Paramecium (Redeker et al., 1994). modifications (PTMs). These PTMs fall into two categories. It is also present in bull sperm flagella (Rüdiger et al., 1995a) Some, such as the acetylation of lysine 40 in certain α-tubulins and echinoderm sperm axonemes (Mary et al., 1996; Multigner (LeDizet and Piperno, 1987) and the phosphorylation sites in et al., 1996) and has recently been found in the flagellated the carboxy-terminal region (see for instance Alexander et al., diplomonad Giardia lamblia (Weber et al., 1996), one of the 1991; Rüdiger and Weber, 1993) are more general PTMs oldest eukaryotes (Sogin et al., 1989). observed also in other proteins. Other PTMs seem to be Trypanosoma are members of the Kinetoplastida, which are tubulin-specific and occur in the acidic carboxy-terminal considered to be more primitive and older eukaryotes than the region. The terminal tyrosine of certain α-tubulins participates ciliates (Sogin et al., 1989; Cavalier-Smith, 1993). They have in a detyrosination/tyrosination cycle, which involves a car- a remarkably simple cytoskeleton which, unlike in other cells boxypeptidase-like activity and the well characterized tubulin- is based primarily on one filament system, the microtubules. tyrosine ligase (Raybin and Flavin, 1975; Ersfeld et al., 1993; Trypanosomal microtubules are found in two major organiz- Thompson, 1982), which restores the tyrosine in an ATP- ations: a subpellicular cage of singlet microtubules, which are dependent reaction. Once α-tubulins have lost also the penul- in close contact with the cell membrane, and the flagellar timate glutamic acid residue they are no longer a substrate for axoneme. This unique organization and the fact that tubulins the ligase (Paturle et al., 1989; Paturle-Lafanechère et al., 1991; are the most abundant proteins in these cells has attracted some Rüdiger et al., 1994). Two other PTMs unique to certain interest in T. brucei as a model system to study a prototype tubulins were established by mass spectrometry of the carboxy- microtubular cytoskeleton. The trypanosomal cytoskeleton has terminal peptides. They involve a polyglutamyl or polyglycyl been characterized in considerable detail on the ultrastructural, sidechain of variable length attached via an isopeptide bond to the biochemical as well as on the genetic level (for review see the γ-carboxylate of a particular glutamic acid residue of α- Seebeck et al., 1990). 432 A. Schneider, U. Plessmann and K. Weber Trypanosomes were the first protozoan organisms for which synthetic peptides. Major peptide peaks (see Results) were also a functional detyrosination/tyrosination cycle of α-tubulin was analyzed by automated Edman degradation using instruments with documented by labelling experiments performed under con- online phenylthiohydantoin amino acid analysis. ditions in which protein synthesis was inhibited (Stieger et al., α Identification of N-acetyl lysine at position 40 of 1984). Similar experiments showed the presence of -tubulin α acetylation (Schneider et al., 1987). Here we used protein flagellar -tubulin chemistry and mass spectrometry of the carboxy-terminal Since lysine 40, the acetylation site of Chlamydomonas and α tubulin peptides together with in vivo labelling experiments to mammalian brain -tubulins (LeDizet and Piperno, 1987; Eddé et al., analyse whether glutamylation and glycylation is already 1990), is situated very early in the largest CNBr fragment it is easy to monitor this residue by Edman degradation (Rüdiger and Weber, present in T. brucei, one of the earliest known eukaryotes. 1993). The CNBr fragments of Trypanosoma flagellar α-tubulin were separated by SDS-PAGE and electrophoretically blotted on to a poly(vinylidenedifluoride) membrane. The largest CNBr fragment MATERIALS AND METHODS corresponding to residues 37 to 203 (Kimmel et al., 1985) was subjected to automated sequencing, which resolves the phenylthiohy- Cells dantoin derivatives of lysine and N-ε-acetyl-lysine (LeDizet and Procyclic T. brucei, stock 427, were grown at 27°C in SDM-79 Piperno, 1987). medium supplemented with 5% fetal bovine serum. Cells were harvested at late log phase corresponding to 1.5×107 to 2.5×107 In vivo labelling with radioactive amino acids cells/ml. Labelling conditions for [3H]glutamate and [3H]glycine were essen- tially as described for [3H]tyrosine (Stieger et al., 1984). For each Cytoskeletal and flagellar fractions labelling experiment, 2×108 exponentially growing (0.5-1.0×107 Detergent extraction of total cytoskeleton and isolation of flagella was cells/ml) trypanosome cells were used. Cells were harvested, washed as described (Schneider et al., 1987). From 2×109 cells approximately once and resuspended in 1/10 of the original volume of HHP-84 2.5 mg of cytoskeletal or 0.5 mg of flagellar proteins were obtained. medium containing all amino acids except the one corresponding to Both fractions were dissolved in SDS sample buffer, boiled and 2 mg the radioactive label (Seebeck and Kurath, 1985). Each of the three each was subjected to SDS-PAGE using a preparative 10% gel. After cultures was split in two aliquots and incubated at 27°C for 30 minutes staining with Coomassie brilliant blue the major band corresponding without and in the presence of protein biosynthesis inhibitors (50 to α- and β-tubulin was excised, washed with water and frozen at µg/ml of cycloheximide, 25 µg/ml each of puromycin and chloram- −70°C until use. phenicol). Subsequently 25 µCi each of [3H]tyrosine (spec. act. 52 Ci/mmol), [3H]glutamate (spec. act. 41 Ci/mmol), or [3H]glycine Isolation and characterization of carboxy-terminal (spec. act. 51 Ci/mmol) were added and incubation was continued for fragments 2 hours. Cells of all six cultures were harvested, washed in HHP-84 medium and dissolved in 100 µl SDS sample buffer. Samples were The α-tubulin fragment was obtained by digestion with endopro- boiled and 50 µl each (approximately 50 µg of proteins) were teinase Lys C and expected to provide residues 431 to 451 in the analysed by SDS-PAGE on a 10% gel. Gels were fixed, incubated for protein sequence predicted by Kimmel et al. (1985). Tubulin present 30 minutes in 1 M sodium salicylate and processed for fluorography. in dye stained gel fragments was electrophoretically concentrated into Dried gels were exposed on preflashed X-ray film (X-omat AR film, a thin band by the agarose gel concentration system (Multigner et al., Kodak SA) for 1 to 40 days depending on the amino acid. Radioac- 1996; Rider et al., 1995). Lys C (Boehringer, Mannheim, Germany) tive amino acids were from Dupont NEN. was used at 3 µg/ml for 16 hours at 37°C in 0.1 M Tris-HCl, pH 8.5, 5% in acetonitrile. The digest was recovered in 20 mM Na-phosphate, pH 7.0 (buffer A) using a fast desalting column and the SMART system (Pharmacia, Uppsala, Sweden). It was separated on a Mono Q RESULTS column (1.6 mm × 50

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